Human herpeviruses have been known to be associated with oncogenic diseases. We previously reported that two regions of HCMV strain Towne can neoplastically transform NIH 3T3 and Rat-2 cells. These are XbaI/BamHI EM (mtrII) and XbaI/BamHI EJ (mtrIII). The mtrII region contains three open reading frames (ORF) of 79, 83 and 34 amino acids. In the current study, we primarily focused on the functional analysis of the mtrII sequence and wondered if mtrII ORFs are responsible for transformation. We generated stable mtrII transformed cell lines with neomycin resistant marker by DNA transfection into NIH 3T3 cells. These cell lines produced tumors in mice. By Northern blot analysis, we consistently detected mtrII specific RNAs in these transformants. We then asked if mtrII contains any promoter sequence for these transcripts. Using chloramphenicol acetyl transferase (CAT) assays in Cos-7 cells, we detected weak promoter activity in the upstream 285 bp region of mtrII when linked to CAT gene in the sense orientation with respect to ORFS. However, the entire 980 bp mtrII region had no detectable promoter activity regardless of the orientation. The 285 bp region also had weak transcriptional enhancer activity only in the sense orientation. Similar results were obtained with CV-1 cells. These studies establish that DNA sequences in mtrII can cis-activate gene expression and are in a location to regulate the expression of the mtrII ORFS. We then examined whether the CMV immediate-early (IE) genes and the HIV TAT gene can regulate the promoter activity in mtrII. No transactivation of the promoter sequence was detected by HIV TAT gene or CMV IE genes. These studies indicate that cellular transcription factors, in large part, control mtrII promoter activity. Further studies would resolve the origin of these transcripts and assess the role of the individual ORFs in mtrII mediated transformation. These studies would be useful to the development of a safe candidate vaccine against human CMV. The project is still active.